The overall goal of this application is to dissect the functional roles of the recently identified B7/CD28 family members ICOS and PD-L1 in regulating mucosal tolerance. This focus is driven by our recent studies that have identified essential, non-redundant roles for ICOS and PD-L1 in mucosal tolerance. We have found that ICOS and PD-L1 have similar roles in controlling the ability of orally-induced regulatory T cells to exert their protective effects. Each of these roles must be unique, since orally induced WT CD4+ Treg cannot function following transfer into ICOS-/- or PD-L1-/- mice. In contrast, PD-L1, but not ICOS, is required for the generation of functional orally induced CD4+ Treg. To investigate the relationships between ICOS and PD- L1 in regulating mucosal tolerance, we will analyze the 1) role of ICOS in the generation and/or function of pathogenic effector T cells (IL-12 driven IFN-g producing Th1 cells and IL-23 driven ThlL-17 cells), since the impaired function of orally induced WT CD4+ T reg could be a primary defect or secondary to enhanced pathogenicity of ICOS-/- effector cells 2) role of ICOS in controlling function of orally induced CD4+ Treg; and 3) relationships between ICOS and PD-L1 in controlling functions of self reactive effector and orally induced CD4+ T reg. These studies should reveal the relative roles of ICOS and PD-L1 in controlling the balance between regulatory and encephalitogenic Th1 cells in vivo. We have assembled a number of novel tools that will enable us to address these issues. To dissect the role of ICOS in mucosal tolerance, we have developed ICOS-/- mice. Our PD-L1-/- mice will serve as a definitive tool to examine how PD-L1 regulates the generation and function of orally-induced CD4+ Treg. We will use MOG35-55 specific TCR transgenic mice to visualize the effects of ICOS and PD-L1 dysregulation on activation, migration and expansion of naive and effector T cells in vivo, and development and behavior of orally induced Treg. These studies should lead to insights into how ICOS and PD-L1 regulate the responses of self-reactive T cells.